In vivo CTL elicitation by heat shock protein fusion proteins maps to a discrete domain and is CD4+ T cell-independent

ABSTRACT

The present invention relates to a method of inducing a CD8 +  CTL response to a molecule in an individual deficient in CD4 +  T cells comprising administering to the individual an hsp or a portion of an ATP binding domain of an hsp joined to the molecule. In one embodiment, the present invention relates to a method of treating HIV in an individual deficient in CD4 +  T cells comprising administering to the individual an hsp or a portion of an ATP binding domain of an hsp joined to the molecule. Also encompassed by the present invention is a method of inducing a CD4 +  independent CTL response in an individual comprising administering to the individual a portion of an ATP binding domain of an hsp joined to the molecule. The present invention also relates to a method of inducing a CD8 +  CTL response in an individual comprising administering to the individual a portion of an ATP binding domain of an hsp joined to the molecule. In addition, the present invention relates to a composition characterized by a portion of an ATP biding domain of an hsp joined to a molecule.

RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.09/761,534, filed on Jan. 16, 2001, which is a continuation ofInternational Application No. PCT/US00/3283 1, which designated theUnited States and was filed on Dec. 1, 2000, which is published inEnglish, and which claims the benefit of U.S. Provisional ApplicationNo. 60/176,143, filed on Jan. 14, 2000. The entire teachings of theabove applications are incorporated herein by reference.

GOVERNMENT SUPPORT

The invention was supported, in part, by National Institutes of Health(NIH) training grant 5T32-AI-07463, NIH Cancer Center core grantCA-14051 and NIH research grants AI44476 and AI44478. The Government hascertain rights in the invention.

BACKGROUND OF THE INVENTION

When injected into an individual with diverse adjuvants, proteinantigens usually stimulate the production of high-affinity IgGantibodies, indicating that they activate CD4 T helper cells, as well asB cells. These procedures generally fail, however, to elicit effectiveCD8 T cell responses. The reason, according to current views, is thatthe short peptides needed, in association with MHC class I molecules, tostimulate CD8 T cells arise from proteolytic cleavage of cytosolicproteins. Since injected protein antigens are generally unable to crosscellular lipid membranes, they fail to gain entry to the propercytosolic “MHC class I processing pathway” and are thus unable tostimulate the production of CD8 T cells. Although there is evidence foralternative cellular pathways for processing some exogenous proteins toform peptide MHC class I complexes (Sigal, L. J., et al., Nature,398:77-80 (1999) and Gromme, M., et al., Proc. Natl. Acad. Sci. USA,96:10326-10331 (1999)) it remains generally true that protein antigensnormally fail to stimulate significant CD8 CTL responses (Rock, K.,Today, 17:131-137 (1996)).

There is now substantial evidence that heat shock proteins (hsps)isolated from tumors can be used as adjuvant-free anti-tumor vaccines inanimals; hsp70 and the distantly related chaperones gp96 andcalreticulin share this immunostimulatory activity (Udono, H. andSrivastava, P. K., J. Exp. Med., 178:1391-1396 (1993); Udono, H., etal., Proc. Natl. Acad. Sci. USA, 91:3077-3081 (1994); Suto, R. andSrivastava, P. K., Science, 269:1585-1588 (1995); Blanchere, N. E., etal., J. Exp. Med., 186:1315-1322 (1997); Tamura, Y., et al., Science,278:117-120 (1997) and Nair, S., et al., J. Immunol., 162:6426-6432(1999)). The fusion of large polypeptides (80-110 amino acids in length)to mycobacterial hsp70 (TBhsp70) creates potent immunogens that canelicit MHC class I-restricted, CD8⁺ cytotoxic T cell responsessufficient to mediate rejection of tumors expressing the fusion partner(Suzue, K., et al., Proc. Natl. Acad. Sci. USA, 94:13146-13151 (1997)).

The means by which soluble hsp70 fusion proteins stimulate CD8 cytotoxicT cell (CTL) responses are unknown. Among the possible mechanismsare: 1) strong hsp-specific CD4⁺ helper cell responses that enhance whatmight otherwise be a minimal response to the soluble proteins (Barrios,C., et al., Eur. J. Immunol., 22:1365-1372 (1992); Suzue, K. and Young,R. A., J. Immunol., 156:873-876 (1996); Horwitz, M. S., et al., NatureMed., 4:781-785 (1998) and Könen-Waisman, S., et al., J. Infect. Dis.,179:403-413 (1999)); and 2) chaperone function of hsps delivers thefusion protein to intracellular compartments of antigen-presenting cellsfor processing into short peptides and loading onto MHC class I (Young,R. A., Ann. Rev. Immunol., 8:401-420 (1990) and Schild, H., et al.,Curr. Opinion Imm., 11:109-113 (1999)). An understanding of the abilityof hsp70 to stimulate CD8⁺ CTL responses is needed to provide for moreeffective immunological prophylaxis and therapy for cancer andinfectious diseases caused by intracellular pathogens.

SUMMARY OF THE INVENTION

The present invention is based on the discovery that a heat shockprotein (hsp; hsps are also known in the art as stress proteins), or adiscrete domain thereof, that is joined to a heterologous molecule canproduce a CD8⁺ cytotoxic (cytolytic) lymphocyte (CTL) response in a hostto which it is administered. The domain can be, for example, about half(e.g., 40, 45, 50, 55, or 60%) of the adenosinetriphosphate (ATP)binding domain of an hsp. Moreover, the response is independent of CD4⁺CTLs. Accordingly, the invention features compositions that include anhsp, or all or a portion of an hsp ATP binding domain, joined to aheterologous molecule and methods of inducing a CD8⁺ CTL response to amolecule in an individual (e.g., a patient, such as a human patient, whohas a deficiency of CD4⁺ T cells) by administering that composition tothe individual. The method can be used to treat a patient who has anacquired immune deficiency syndrome (AIDS) by, for example,administering to the patient an hsp, or a portion of an ATP bindingdomain of an hsp, that is joined to a molecule associated with a humanimmunodeficiency virus (HIV), such as an HIV antigen.

The invention has numerous advantages. For example, the compositions andmethods described herein provide for highly effective CD8⁺ CTLresponses. These responses are useful in treating (i.e., preventing orreducing the length or severity of symptoms associated with a diseaseprocess or preventing or attenuating the cellular events through whichthose symptoms are made manifest; treatment may be effective withoutcompletely eradicating all symptoms) diseases that are caused by orotherwise associated with intracellular pathogens. Diseases orconditions that are characterized by a deficiency (or complete lack of)CD4⁺ T cells are particularly amenable to treatment.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A is a graph of effector cell to target cell ratio (E:T) versus %specific lysis illustrating OVA-specific CTL elicited by immunizationwith OVA.TBhsp70 fusion protein without adjuvant in the splenocytes fromwild-type C57/BL/6 mice.

FIG. 1B is a graph of E:T versus % specific lysis illustratingOVA-specific CTL elicited by immunization with OVA.TBhsp70 fusionprotein without adjuvant in the splenocytes from CD4⁺ knockout mice (CD4−/− mice).

FIG. 1C is a graph of E:T versus % specific lysis illustratingOVA-specific CTL elicited by immunization with OVA.TBhsp70 fusionprotein without adjuvant in the splenocytes from mice which have veryfew CD8⁺ T cells (β2m −/− mice).

FIG. 2A is a graph of E:T versus % specific lysis illustrating murinehsp70 fusion protein elicits CTL responses in wild-type C57BL/6 mice.

FIG. 2B is a graph of E:T versus % specific lysis illustrating murinehsp70 fusion protein elicits CTL responses in CD4 −/− mice.

FIG. 3 is an illustration of the domains of the full length TBhsp70which were separated into four segments I, II, III and IV and fused toC-terminal of OVA to make OVA.TBhsp70 fusion proteins; the numbersbeneath each segment refer to the amino acid positions in TBhsp70.

FIG. 4 is a graph of E:T versus % specific lysis illustratingOVA-specific T cell responses in mice immunized with OVA fused todomains of TBhsp70; splenocyte cultures from mice primed with OVA (Δ),OVA.TBhsp70 (▪), OVA.TBhsp70 I (∇), II (♦), III (X) and IV (+) were usedas effector cells in the cytotoxicity assay.

FIG. 5A illustrates the P1 peptide amino acid sequence (SEQ ID NO: 25),aligned over a diagram of the hsp65-P1 fusion protein (P1 is shown atthe C-terminal of hsp65). When liberated from P1, SIYRYYGL (SEQ IDNO: 1) (demarked by arrows) binds to K^(b) to form the peptide-MHCcomplex recognized by the 2C TCR. In P1, SIYRYYGL (SEQ ID NO: 1) isflanked 5′ and 3′ by sequences that lie immediately upstream anddownstream, respectively, of peptide bonds that are cleaved (see arrows)in murine cells to liberate naturally occurring peptides (SIINFEKL (SEQID NO: 2) from ovalbumin (Ova) and LSPFPFDL (SEQ ID NO: 3) fromα-ketoglutaraldehyde dehydrogenase (αKG) (Falk, K., et al., Eur. J.Immunol., 22:1323-1326 (1992); Ukada, K., et al., J. Immunol.,157:670-678 (1996)).

FIG. 5B is pair of histograms, which display experimental evidence thatP1 and hsp65-P1 are processed intracellularly to yield the SYRGL (SEQ IDNO: 4) octapeptide. 48 hr after transfection with mammalian expressionvectors (VR1055 and pCINeo), containing sequences that encode P1 andhsp65-P1, respectively, EL4 cells were incubated for 18 hr with an equalnumber of naive 2C T cells. Histograms show the percentage of live,2C⁺CD8⁺ cells that were stimulated to upregulate the activation markerCD69. The responses of these naive T cells to control EL4 cells,transfected with the empty (vector) plasmids, are shown as shadedhistograms.

FIG. 5C is a graph which displays experimental evidence that normalC57BL/6 mice have T cells that can recognize the SYRGL-K^(b) complex. ACD8⁺ T cell line, derived from C57BL/6 mice immunized with the SYRGL(SEQ ID NO: 4) peptide in adjuvant, specifically lysed T2-K^(b) targetcells in a peptide-dependent manner. A highly cytolytic long-termcultured 2C CTL clone (L3.100) is shown for comparison.

FIG. 6A is a graph showing CD8⁺ CTL that recognize the SYRGL-K^(b)complex are produced in C57BL/6 mice injected with hsp65-P1 in PBS butnot in those injected similarly with equimolar amounts of variouscontrols (a mixture of P1 and hsp65, the SYRGL (SEQ ID NO: 4)octapeptide, the P1 polypeptide itself, or hsp65 itself; as notedfurther below, SYRGL is referred to as an “octapeptide” as it is anabbreviation of the sequence SIYRYYGL (SEQ ID NO.: 1)).

FIG. 6B is a graph illustrating the production of SYRGL-specific CTL inmice injected with various amounts of hsp65-P1, 0.015-1.5 nmoles (1-100μg) or a control fusion protein in which P1 is linked to the C-terminusof a maltose-binding protein (Ma1-P1, 80 μg); lysis of T2-K^(b) targetcells in the absence of added SYRGL (SEQ ID NO: 4) peptide is indicatedby unfilled symbols.

FIG. 6C are graphs showing depletion of CD8⁺ T cells eliminates theSYRGL-specific CTL produced by mice injected with hsp65-P1. Lymph nodeand spleen cells from C57B/6 mice immunized with 1.5 nmoles of hsp65-P1or Ma1-P1 were cultured for 6 days and then depleted of CD8 T cells bymagnetic sorting. The untreated, CD8-depleted, and CD8-enrichedpopulations (30%, 1%, 90%, CD8⁺ T cells respectively) were analyzed in a4 hr cytolytic assay; lysis of T2-K^(b) target cells in the absence ofadded SYRGL peptide is indicated by unfilled symbols.

FIG. 7 is a graph showing ⁵¹Cr-labeled splenic dendritic cells (sp1 dc),bone-marrow derived dendritic cells (bm-dc), or purified macrophages(mø), isolated from peritoneal lavage, all from B6 (H-2^(b)) mice wereincubated for 4 hr with a 2C CTL clone (L3.100: see FIG. 5D) and variousconcentrations of the SYRGL octapeptide. CTL target cell ratio(E:T)=5:1. Unfilled symbols show lysis when the control fusion protein(Ma1-P1) was used in place of hsp65-P1.

FIG. 8A illustrates that splenic dendritic cells and peritoneal lavagemacrophages were purified by magnetic sorting and incubated for 18-24 hrwith equimolar concentrations of hsp65-P1 or Ma1-P1 before adding naive2C T cells. Expression of the activation marker CD69, Hsp65-P1 or Ma1-P1were added to purified splenic dendritic cells, macrophages, or to mediaalone (“no APC”) at 15 nM (˜1 μg/ml). After 24 hr, purified naive 2C Tcells were added (T cell:APC ratio of 1:1), and 18 hr later cells wereanalyzed for CD69, gating on propidium iodide-negative 2C⁺CD8⁺ cells.The percentage of 2C T cells with increased expression of CD69⁺ isindicated.

FIG. 8B illustrates that splenic dendritic cells and peritoneal lavagemacrophages were purified by magnetic sorting and incubated for 18-24 hrwith equimolar concentrations of hsp65-P1 or unmodified hsp65 beforeadding naive 2C T cells. Dendritic cells or macrophages were incubatedwith hsp65-P1 or hsp65 before adding the naive 2C T cells and incubationwas continued for an additional 18 hr (IL-2 assay) or 60 hr(proliferation assay) or 48 hr (IFN-γ assay). “No Ag” means thedendritic cells and 2C T cells were present but hsp65-P1 and hsp65 wereabsent: “No T” cells means the hsp65-P1 was present but the 2C T cellswere omitted.

FIG. 8C is a graph illustrating inhibition of responses by a clonotypicmonoclonal antibody to the 2C TCR (1B2). Bone marrow derived dendriticcells were incubated with 10 μg/ml hsp65-P1 overnight. Equal numbers ofnaive 2C T cells were then added in the presence or absence of 1B2 Fabfragments (25 μg/ml). After an additional 18 hr, cells and supernatantswere analyzed, respectively, for CD69 expression (left panel) and IL-2production (3H-thymidine incorporation by IL-2-responsive HT2 cells,right panel).

FIG. 9A is a pair of graphs comparing dendritic cells' and macrophages'ability to stimulate T cell responses at limiting antigen dose in vitro.Fresh splenic dendritic cells or macrophages were incubated with variousconcentrations of hsp65-P1 or Ma1-P1 fusion proteins for about 18 hrbefore adding purified naive 2C T cells (see FIGS. 8A, 8B). Supernatantswere sampled 18 hr later to determine IL-2 levels (upper panel).³H-thymidine was added at 48 hr and cells were harvested after anadditional 18 hr to assess T cell proliferation (lower panel).

FIG. 9B is a pair of graphs illustrating the behavior of hsp65 fusionprotein-activated dendritic cells in vivo. Myeloid dendritic cells fromlymph nodes draining a subcutaneous site where hsp65-P1 was injected 24hr previously show increased expression of MHC-1 (K^(b)) (lower panel)compared to myeloid dendritic cells from lymph nodes draining anuninjected site (“no treatment”, upper panel).

FIG. 9C is a trio of graphs illustrating the behavior of hsp65 fusionprotein-activated dendritic cells in vivo. Dendritic cells activatedwith a noncognate hsp fusion protein (hsp65-NP) and pulsed with 10⁻⁹ MSYRGL (SEQ ID NO: 4) peptide are more effective than nonactivated,similarly pulsed dendritic cells in stimulating naive T cells in vivo.8×10⁵ dendritic cells were injected into a hind footpad of normal B6mice that had been injected (iv) with 2×10⁶ naive 2C TCR+ cells (from 2CTCR transgenic mice RAG-deficient mice). 24 hrs after the footpadinjection, 2C CD8⁺ T cells in the draining popliteal lymph node wereexamined for CD69 expression. Frequency of CD69⁺ 2C CD8⁺ T cells in alymph node draining the site where activated (control) dendritic cells(not pulsed with peptide) were injected (upper panel), or where SYRGLpeptide-pulsed (1×10⁻⁹M) unactivated dendritic cells or activateddendritic cells were injected (middle panel and lower panel,respectively). Percentages of CD69⁺ 2C cells are shown. Geometric meansfluorescence values for MHC-1 (K^(b)) on dendritic cells that had beenincubated, prior to footpad injection, with or without hsp65-NP were 379and 97, respectively.

FIG. 10A is a pair of graphs of dendritic cell MHC class I expressionplotted as a function of protein concentration of the added hsp fusionproteins and control proteins. Upper panel; dendritic cells from C57BL/6mice. Lower panel; dendritic cells from C3H mice. Purified bone marrowderived dendritic cells were incubated for 24 hr with variousconcentrations of hsp65-P1 or other hsp65 fusion proteins, having asfusion partners influenza virus nucleoprotein (hsp65-NP) or humanpapilloma virus, type 16, E7 subunit (hsp65-E7 preparations #1 and #2)or with controls (hsp65 alone, P1 alone, E7 alone, an anti-TNP IgGantibody). MHC Class I protein levels on the dendritic cells were thendetermined by flow cytometry by gating on propidium iodide-negativeCD11c⁺ cells and using the Y3 antibody which recognizes both H-2^(b)(K^(b)) and H-2^(k) MHC class I. MHC class I levels are shown asgeometric mean fluorescence; the levels on untreated dendritic cells arerepresented by a dashed horizontal line.

FIG. 10B is a pair of graphs of the dendritic cell MHC class Iexpression values from FIG. 10A and are plotted as a function ofendotoxin concentration (calculated from the endotoxin levels present inthe added hsp fusion proteins and other proteins). Upper panel;dendritic cells from C57BL/6 mice. Lower panel: dendritic cells from C3Hmice.

FIG. 10C is a graph showing that Hsp65-P1 stimulates production of CTL(anti-SYRGL) in CD4-deficient (CD4^(−/−)) mice. As in FIG. 6A-6C themice were injected s.c. twice, one wk apart, with 100 μg of hsp65-P1 orMa1-P1 in PBS. One wk following the second injection, cells from spleenand draining lymph nodes were pooled and restimulated with 1 μM SYRGL(SEQ ID NO: 4) peptide without addition of exogenous cytokines. Six dayslater the cells were used as effectors in a standard 4 hr cytolyticassay at various E:T ratios using ⁵¹Cr-labeled T2-K^(b) cells as targetsin the presence of 1 μM SYRGL (SEQ ID NO: 4). Lysis of T2-K^(b) cells inabsence of SYRGL (SEQ ID NO: 4) is shown by unfilled symbols.

FIG. 11 is the nucleotide (cDNA) (SEQ ID NO: 5) and amino acid (SEQ IDNO: 6) sequences of Mycobacterium tuberculosis hsp70 (TBhsp70) whereinsegment II (nucleotides 481-1110; amino acids 161-370) is highlighted.

FIG. 12 is the nucleotide (SEQ ID NO: 7) and amino acid (SEQ ID NO: 8)sequences of segment II of TBhsp70.

FIGS. 13A-13B are the nucleotide (SEQ ID NO: 9) and amino acid (SEQ IDNO: 10) sequences of murine hsp70 wherein segment II (nucleotides568-1194; amino acids 190-398) is highlighted.

FIG. 14 is the nucleotide (SEQ ID NO: 11) and amino acid (SEQ ID NO: 12)sequences of segement II of murine hsp70.

DETAILED DESCRIPTION OF THE INVENTION

An immunological response to a molecule that, notably, includes a CD8⁺CTL response, can be evoked in an individual by administering to thatindividual either an hsp joined to that molecule or a portion of an ATPbinding domain of an hsp joined to that molecule (the molecule beingvirtually any biological substance, naturally- or nonnaturally-occurringwith the exception of a portion of a stress protein). The CD8⁺ CTLresponse can be evoked in an individual who has a deficiency of CD4⁺ Tcells (i.e. a CD4⁺ T cell count considered by any routinely used medicalstandard to be physiologically abnormal). Physicians and others havingordinary skill in the art can identify such individuals, which includepatients infected with HIV. Accordingly, patients who are infected withHIV, or at risk of becoming so, can be treated with either an hsp joinedto an HIV antigen (e.g., p24 or gp41) or a portion of an ATP bindingdomain of an hsp joined to an HIV antigen (e.g., p24 or gp41).Physicians and others having ordinary skill in the art can recognize anduse other molecules associated with the HIV.

Heat shock proteins useful in the present invention are those that havean ATP binding domain and those that, when administered to anindividual, induce a CD8⁺ T cell response to a molecule to which theyare joined. Full length hsps (e.g., hsp70 and hsp65) can be used, as canthe ATP binding domains of hsps (or portions thereof). For example, thehsp moiety joined to the molecule can be the amino-terminal portion ofthe ATP binding domain. For example, the hsp moiety joined to themolecule can include or consist of about half of the ATP binding domain.For example, the hsp moiety can include or consist of about 25 to about365 consecutive amino acid residues (e.g. 25-350, 50-300, 110-275,125-250, 130-225, 135-200, 150-200, 170-190, or 100-200 residues) of theATP binding domain. More specifically, the hsp moiety can include orconsist of amino acid residues 161-370 of Mycobacterium tuberculosishsp70 or amino acid residues 190-398 of murine hsp70. Portions of hsp65that are homologous to segment II of hsp70 (e.g. mycobacterial hsp65such as Mycobacterium bovis BCG; mammalian hsp65, such as murine,canine, porcine, equine or human hsp65) can be used as described herein.

Those of ordinary skill in the art are well able to identify hsps andATP binding domains within those proteins. Moreover, those artisans canmake substitutions, if desired, in the sequences of these proteins ortheir domains that do not substantially reduce the abilities of thoseproteins or their domains to effectively induce CD8⁺ T cell responses.Amino acid substitutions can be made on the basis of similarity inpolarity, charge, solubility, hydrophobicity, hydrophilicity, or theamphipathic nature of the residues involved. For example, the nonpolar(hydrophobic) amino acid residues alanine, leucine, isoleucine, valine,proline, phenylalanine, tryptophan, and methionine can be substitutedone for another; polar neutral amino acid residues such as glycine,serine, threonine, cysteine, tyrosine, asparagine, and glutamine can besubstituted one for another; positively charged (basic) amino acidresidues such as arginine, lysine, and histidine can be substituted onefor another; and negatively charged (acidic) amino acid residues such asaspartic acid and glutamic acid can be substituted one for another. Forexample, the hsp moiety used as described herein can include 1-25%conservative amino acid substitutions.

Any hsp or any portion of the hsp ATP binding domain can be purifiedfrom natural sources, recombinantly produced, or chemically synthesized.For example, an hsp or a portion thereof can be obtained frommycobacteria (e.g., Mycobacterium tuberculosis, Mycobacterium bovis,Mycobacterium leprae, or Mycobacterium smegmatis), mammals (e.g. amurine, canine, porcine, equine, or human), fungi, parasite, orbacteria. Methods for recombinantly producing hsps or portions thereofare also well known (production in bacteria such as E. coli) aredescribed herein. In addition, the hsp or the portion thereof can beobtained from a commercial supplier.

Molecules useful in the present methods include any molecule againstwhich a CD4⁺ independent immune response is desired. A “molecule”includes, but is not limited to, proteins or fragments thereof (e.g.,proteolytic fragments), peptides (e.g., synthetic peptides orpolypeptides), antigens, glycoproteins, carbohydrates (e.g.,polysaccharides and oligosaccharides), lipids, glycolipids, DNA (e.g.,recombinant DNA), killed or attenuated whole organisms (e.g., viruses,bacteria, mycobacteria, parasites or fungi) or portions thereof, toxins,toxoids or any other organic molecule.

Molecules useful in the present methods can be obtained from a varietyof sources using techniques routinely practiced in the art. For example,the molecule can be obtained from pathogens or organisms such asbacteria, mycobacteria, viruses, fungi or parasites. While the moleculecan be isolated (e.g., purified or partially purified (e.g. physicallyseparated from at least 50% of the biological substances with which itnaturally associates), it can also be chemically synthesized,recombinantly produced, or purchased from a commercial supplier.

The hsp or portion thereof is “joined” to a molecule against which animmune response is desired. The term “joined” includes covalentattachment of the hsp, or a portion thereof, to the molecule. Theconjugation can be carried out using techniques routinely practiced inthe art (e.g., by forming a covalent bond between the hsp, or theportion thereof, and the molecule or by reductive amination). The term“joined” also includes fused proteins, such as those created byrecombinant techniques or chemical synthesis. The fusion protein caninclude the molecule fused to the amino-terminal region or theC-terminal region of the hsp, or the portion thereof.

The CD8⁺ CTL responses induced by the methods of the present inventioncan be used for prophylaxis and/or therapy of diseases or conditions,particularly those characterized by a lack or deficiency of CD4⁺ Tcells. That is, the hsp or portion thereof joined to the moleculeagainst which an immune response is desired can be administered to anindividual either before or after a disease or condition is manifestedand can result in prevention, amelioration, elimination or delay in theonset or progression of the disease state. For example, the presentinvention can be used to prevent or treat an individual positive forhuman immunodeficiency virus (HIV) and the opportunistic infectionsassociated with HIV. In one embodiment, the HIV positive individual isdeficient in CD4⁺ T cells.

In the methods of the present invention, an effective amount of the hspor portion thereof joined to the molecule against which an immuneresponse is desired is administered to an individual (e.g., mammal suchas human). As used herein an “effective amount” is an amount thatinduces a CD4⁺ T cell independent immune response to the molecule in anindividual. In a particular embodiment, an “effective amount” is anamount such that when administered to an individual, it results in anenhanced CD8⁺ CTL response to the molecule relative to the CD8⁺ CTLresponse to the molecule in an individual to whom an effective amountwas not administered. For example, an effective amount or dosage of thehsp or portion thereof joined to the molecule against which an immuneresponse is desired is in the range of about 50 pmoles to about 5000pmole. In one embodiment, the dosage range if from about 80 pmole toabout 3500 pmoles; in another embodiment, the dosage range is from about100 pmoles to about 2000 pmoles; and in a further embodiment the dosagerange is from about 120 pmoles to about 1000 pmoles. The appropriatedosage of hsp or portion thereof joined to the molecule against which animmune response is desired for each individual will be determined bytaking into consideration, for example, the particular hsp and/ormolecule being administered, the type of individual to whom thecomposition is being administered, the age and size of the individual,the condition or disease being treated or prevented and the severity ofthe condition or disease. Those skilled in the art will be able todetermine using no more than routine experimentation the appropriatedosage to administer to an individual.

The hsp or portion thereof joined to the molecule against which theimmune response is desired can be administered to the individual in avariety of ways. The routes include intradermal, transdermal, (e.g.,slow release polymers), intramuscular, intraperitoneal, intravenous,subcutaneous, oral, epidural and intranasal routes. Any other convenientroute of administration can be used, for example, infusion or bolusinjection, infusion of multiple injections over time, or absorptionthrough epithelial or mucocutaneous linings. In addition, the hsp joinedto the molecule can be administered with other components orbiologically active agents, such as adjuvants, pharmaceuticallyacceptable surfactants (e.g., glycerides), excipients, (e.g., lactose),liposomes, carriers, diluents and vehicles.

Further, in the embodiment in which the molecule is a protein (peptide),the hsp or portion thereof joined to the molecule can be administered byin vivo expression of polynucleotides encoding such into an individual.For example, the hsp or portion thereof and/or the molecule can beadministered to an individual using a vector, wherein the vector whichincludes the hsp or portion thereof joined to the molecule isadministered under conditions in which the hsp or portion thereof andthe molecule are expressed in vivo.

Several expression system vectors that can be used are availablecommercially or can be produced according to recombinant DNA and cellculture techniques. For example, vector systems such as yeast orvaccinia virus expression systems, or virus vectors can be used in themethods and compositions of the present invention (Kaufman, R. J., J.Meth. Cell and Molec. Biol., 2:221-236 (1990)). Other techniques usingnaked plasmids or DNA, and cloned genes encapsulated in targetedliopsomes or in erythrocyte ghosts can be used to introduce the hsp orportion joined to the molecule into the host (Friedman, T., Science,244:1275-1281 (1991); Rabinovich, N. R., et al., Science, 265:1410-1404(1994)). The construction of expression vectors and the transfer ofvectors and nucleic acids into various host cells can be accomplishedusing genetic engineering techniques, as described in manuals likeMolecular Cloning and Current Protocols in Molecular Biology, which areincorporated by reference, or by using commercially available kits(Sambrook, J. et al., Molecular Cloning, Cold Spring Harbor Press, 1989;Ausubel, F. M., et al., Current Protocols in Molecular Biology, GreenePublishing Associates and Wiley-Interscience, 1989).

As demonstrated in Example 1, hsp70 fusion proteins elicit CD8⁺ CTL inthe absence of CD4⁺ T lymphocytes and this function resides in a200-amino acid segment of TBhsp70, indicating that chaperone activity isnot required. To gain insights into the mechanisms by which soluble hspfusions can elicit CD8⁺ CTL against the fusion partner, hsp70 wasdissected to ascertain whether a particular hsp domain is necessary, andknockout mice were used to determine whether the fusion protein'simmunogenicity is dependent on CD4⁺ T lymphocytes. It was found that theability to elicit CD8⁺ CTL depends on a discrete 200-amino acid proteindomain, indicating that the fusion protein's immunogenicity for CD8⁺ Tcells does not require coupled chaperone function or peptide binding.Further, it was found that ovalbumin.hsp70 fusion protein elicitedanti-ovalbumin CD8⁺ CTL about equally well in CD4 knockout and wild-typeC57BL/6 mice, and also when the hsp70 was of mycobacterial(Mycobacterium tuberculosis) or murine (self) origin. The ability ofhsp70 fusion proteins to elicit CD4-independent CTL responses indicatesthat hsp70 fusion proteins can be used for immunological prophylaxis andtherapy against disease in CD4⁺ T cell deficient individuals.

As demonstrated in Example 2, a mycobacterial heat shock protein, 65 kDa(hsp65), fused to a polypeptide (P1) that contains an octapeptide(SIYRYYGL (SEQ ID NO: 1)) agonist for a particular T cell receptor (2CTCR) stimulated C57BL/6 mice, as well as CD4-deficient mice, to produceCD8⁺ cytolytic T lymphocytes (CTL) to the fusion partner's octapeptide.This and other hsp65 fusion proteins, but not native hsp65 itself,stimulated dendritic cells, in vitro and in vivo, to upregulate thelevels of MHC (class I and II) and costimulatory (B7.2) molecules. Theresults provide a mechanism for the general finding that hsp fusionproteins, having fusion partners of widely differing lengths andsequences, elicit CD8 CTL to peptides from the fusion partners, withoutrequiring exogenous adjuvants or the participation of CD4⁺ T cells.

When mycobacterial hsp fused with large protein fragments, termed fusionpartners, are injected into mice in saline solution (PBS) without addedadjuvants several of them were previously shown to stimulate theproduction of CD8 CTL that recognize short peptide epitopes (8-10 aminoacids in length) that arose from the fusion partners. The fusionpartners varied from about 80 to 110 amino acids in length and werederived from ovalbumin (Suzue, K., et al., Proc. Natl. Acad. Sci, USA,94:13146-13151 (1997)), influenza virus nucleoprotein (Anthony, L., etal., Vaccine, 17:373-383 (1999)), and an entire protein subunit of ahuman papilloma virus (N. R. Chu, personal communication). As describedin Example 2, to explore the mechanisms that permit these hsp to beeffective with such diverse fusion partners, and that enable the hspfusion proteins to serve as effective immunogens for CD8 T cells withoutrequiring adjuvants, the immunogenic activities of fusion proteinsprepared from the 65 kDa hsp from Mycobacterium bovis, BCG strain (herecalled hsp65) were studied.

The principal fusion partner used in Example 2 was a polypeptide thatcontains an octapeptide sequence, SIYRYYGL (SEQ ID NO: 1) (hereaftercalled SYRGL (SEQ ID NO: 4), Udaka, K., et al., Cell, 69:989-998(1992)), which together with K^(b) serves as a potent stimulator of CD8T cells having the TCR of a CTL clone called 2C (Kranz, D., et al.,Proc. Natl. Acad. Sci. USA, 81:573-577 (1984)). This peptide wasidentified in a synthetic peptide library and, so far as is known, doesnot occur in nature. The use of various T cells that express the 2C TCR,particularly naive 2C T cells (Cho, B., et al., Proc. Natl. Acad. Sci.USA, 96:2976-2981 (1999)), were relied on as specific probes to obtainevidence that i) dendritic cells are more effective than macrophages inpresenting the processed hsp fusion protein to naive CD8 T cells, ii)dendritic cells are stimulated directly by each of several hsp65 fusionproteins tested, but not by “native” hsp65 itself, to increase surfaceexpression of MHC class I and II and costimulatory (B7.2) molecules, andiii) CD4 T cells are not required for the fusion protein's ability toelicit production of CD8 CTL in vivo. Taken together, the resultsdescribed herein indicate that diverse soluble heat shock fusionproteins, regardless of the length or sequence of the fusion partners,stimulate CD8 T cell responses to peptides derived from the fusionpartners without requiring exogenous adjuvants. The findings are ofparticular interest in view of the need to develop protective vaccinesagainst intracellular pathogens for which current immunizationstrategies are inadequate (e.g., against HIV-1, human papilloma virus,various herpes viruses, malaria).

EXEMPLIFICATION Example 1 In Vivo CTL Elicitation by hsp70 FusionProteins Maps to a Discrete Domain and is CD4⁺ T Cell-Independent

Materials and Methods

Expression Vectors:

All constructs used to produce OVA.hsp70 fusion proteins were made inthe bacterial expression plasmid pKS11h (Suzue, K., et al., Proc. Natl.Acad. Sci. USA, 94:13146-13151 (1997)). Fusion constructs, consisting ofOVA fused to the N-terminus of various segments of hsp70, were inserteddownstream of the histidine tag sequence. A portion of ovalbumin (aminoacid 230-359, hereafter referred to as OVA) was amplified from pOV230(McReynolds, L. A., et al., Gene, 2:217-231 (1977)) by PCR usingupstream primer oQH025 and the downstream primer oQH027. Functional andstructural domains of TBhsp70 based on crystal structures of ATP domainof bovine hsc70 (Flaherty, K. M., et al., Nature, 346:623-628 (1990))and peptide-binding domain of E. coli DnaK (Zhu, X., et al., Science,272:1606-1614 (1996)) were used. The full-length TBhsp70 were separatedinto four segments I, II, III and IV. The full-length TBhsp70 and eachsegment were fused to C-terminal of OVA to make OVA.TBhsp70 fusionproteins. (The sequences of these and other PCR primers are listed atthe end of the Methods and Materials section).

The OVA expression vector pQH07 was constructed by subdloning OVA intothe NdeI and NheI sites of pKS11h. Full-length TBhsp70 and fourtruncated TBhsp70 segments I (aa 1-166), II (aa 161-370), III (aa360-517) and IV (aa 510-625) were amplified from plasmid pY3111/8 (kindgift of W. Wu, StressGen Biotechnologies, Vancouver Canada). Theupstream primer for full-length TBhsp70 and segment I is oQH001, and thedownstream primers are oJR061 and oQH011, respectively. The upstreamprimers for TBhsp70 II, III and IV are oQH012, oQH014 and oQH106,respectively. The downstream primers are oQH013, oQH015 and oJR061,respectively. The plasmids pQH06, pQH08, pQH09, pQH10 and pQH11, whichexpress OVA fused to TBhsp70, TBhsp70 segment I, segment II, segment IIIand segment IV respectively, were constructed by subdloning thefull-length and truncated TBhsp70 PCR products into the Bam-HI and EcoRIsites of pQH07 (at the C-terminus of OVA). Murine hsp70.1 codingsequence (referred to here as mhsp70) was amplified from plasmidpmhsp70.1 by PCR using the upstream primer oJR102 and the downstreamprimer oJR103. Plasmid pQH12, expressing OVA.mhsp70 fusion protein, wascreated by subcloning mhsp70 into the BamHI and EcoRI sites of pQH07.All plasmids were verified by sequencing in both directions withdouble-stranded DNA templates.

Recombinant Protein Purification:

OVA, OVA.TBhsp70, OVA.TBhsp70 II, OVA.TBhsp70 III, and OVA.TBhsp70 IVwere induced in E. coli (BL21(DE3)pLysS) for 9 hours at 25° in thepresence of 0.5-1 mM isopropyl thiogalactoside (IPTG) and were purifiedas soluble proteins. The mycobacterial segment I and murine hsp70 fusionproteins were induced in E. coli for 4 hours at 37° with 1 mM IPTG andpurified from inclusion bodies and then refolded as previously described(Suzue, K., et al., Proc. Natl. Acad. Sci. USA, 94:13146-13151 (1997)and Suzue, K. and Young, R. A., J. Immunol., 156:873-876 (1996)). Allproteins were purified using nitrilo-triacetic acid Ni+ column (Qiagen,Hilden Germany) and HiTrap-Q anion exchange chromatography (Pharmacia,Piscataway, N.J.) as previously described (Suzue, K., et al., Proc.Natl. Acad. Sci. USA, 94:13146-13151 (1997) and Suzue, K. and Young, R.A., J. Immunol., 156:873-876 (1996)). Purity was assessed using 4-20%gradient SDS-PAGE gels stained with Coomassie Blue (Bio-Rad, HerculesCalif.). All proteins were dialyzed against phosphate-buffered saline(PBS), and sterile filtered at 0.2 μM. Protein concentrations weremeasured by Lowry assay (Bio-Rad) and expressed in molar terms to allowsimple comparison of proteins of differing molecular weights.

Mice and Immunizations:

Six- to eight-week old female C57BL/6, CD4−/− and β2m−/− mice wereobtained from the Jackson Laboratory (Bar Harbor, Me.) and Taconic Farms(Germantown, N.Y.). Both knockout mice have C57BL/6 (H-2^(b)) geneticbackgrounds. Groups of 3 to 4 mice were injected intraperitoneally(i.p.) with 120 pmoles of recombinant protein in PBS; a second injectionwas performed subcutaneously (s.c.) two weeks later. The mice weresacrificed 10 days after the boost and splenocytes within groups werepooled (Suzue, K., et al., Proc. Natl. Acad. Sci. USA, 94:13146-13151(1997)).

Cell Line:

EG7-OVA cells were cultured as previously described (Suzue, K., et al.,Proc. Natl. Acad. Sci. USA, 94:13146-13151 (1997)). OVA-specific CTLelicited by immunization with OVA.TBhsp70 fusion protein withoutadjuvant were examined. The splenocytes from mice immunized with OVA (Δ)or OVA.TBhsp70 (▪) were incubated with irradiated EG7-OVA cells for 6days in the absence of added cytokines and then used as effector cells(E) in a standard 4 hour cytotoxicity assay. The ⁵¹Cr-labeled targetcells (T) were: T2-K^(b) (dashed line) and T2-K^(b)-pulsed with SIINFEKLpeptide (SEQ ID NO: 2) (solid line) at 33 μg/ml. Splenocytes fromwild-type C57/BL/6 mice are shown in FIG. 1A; splenocytes from CD4 −/−mice are shown in FIG. 1B; and splenocytes from β2m −/− mice are shownin FIG. 1C.

CTL Assays:

CTL assays were performed as described (Suzue, K., et al., Proc. Natl.Acad. Sci. USA, 94:13146-13151 (1997)). Splenocyte cultures from miceprimed with OVA (Δ), OVA.TBhsp70 (▪), OVA.TBhsp70 I (□), II (♦), III (X)and IV (+) were used as effector cells in the cytotoxicity assay (SeeFIG. 4). Results shown are representative of experiments repeated two tofive times. PCR primers: oQH025 (5′-GCAGTACTCATATGATCCTGGAGCTTCCA (SEQID NO: 13) TTTGCCAGTGGGACAATG-3′) oQH027(5′-CTCCGACCTCACCTACGACGTTCGCAGAG (SEQ ID NO: 14)ACTTCTTAAAATTATCCGATCGCCTAGACCTAG T-3′) oQH001(5′-ATAGTACTGGATCCATGGCTCGTGCGGTC (SEQ ID NO: 15) GGGATCGACCTCGGG-3′)oJR061 (5′-GGAATTCCTATCTAGTCACTTGCCCTCCC (SEQ ID NO: 16) GGCCGTC-3′)oQH011 (5′-GTCGACGAATTCATCATCAGATTCGCTGC (SEQ ID NO: 17)TCCTTCTCGCCCTTGTCGAG-3′) oQH012 (5′-GTCGACGGATCCATGGAGAAGGAGCAGCG (SEQID NO: 18) AATCCTGGTCTTCGACTTG-3′) oQH014(5′-GTCGACGGATCCATGGTGAAAGACGTTCT (SEQ ID NO: 19)GCTGCTTGATGTTACCCCG-3′) oQH016 (5′-GTCGACGGATCCATGCGTAATCAAGCCGA (SEQ IDNO: 20) GACATTGGTCTACCAGACG-3′) oQH013 (5′-GTCGACGAATTCATCACGGGGTAACATCA(SEQ ID NO: 21) AGCAGCAGAACGTCTTTCAC-3′) oQH015(5′-GTCGACGAATTCATCAGACCAATGTCTCG (SEQ ID NO: 22)GCTTGATTACGAACATCGGC-3′) oJR102 (5′-TCTAGAGGATCCATGGCCAAGAACACGGC (SEQID NO: 23) GATC-3′) oJR103 (5′-TCTAGAGAATTCCTAATCCACCTCCTCGA (SEQ ID NO:24) TGGTGGGTCC-3′)

Results and Discussion

Previous studies demonstrated that soluble, adjuvant-free TBhsp70 fusionproteins elicit substantial immune responses, including CD8⁺ CTLs, inmice (Suzue, K., et al., Proc. Natl. Acad. Sci. USA, 94:13146-13151(1997) and Suzue, K. and Young, R. A., J. Immunol, 156:873-876 (1996)).The basis for the effectiveness of hsp70 fusions is unclear as mostsoluble proteins do not elicit significant CD8⁺ T cell responses(reviewed in Braciale, T. J., et al., Immunol. Rev., 98:95-114 (1987),Jondal, M., et al., Immunity, 5:295-302 (1996)). While there is evidencethat the hsp moiety of mycobacterial hsp fusion proteins acts as aneffective carrier in the classic sense, enhancing B cell responses tochemically conjugated pneumococcal polysaccharides (Könen-Waisman, S.,et al., J. Infect. Dis., 179:403-413 (1999)) and malarial polypeptide(Barrios, C., et al., Eur. J. Immunol., 22:1365-1372 (1992)), carriersare not known to stimulate CTL production. It was reasonable to expectthat hsp70 fusion proteins provide hsp70-specific cognate CD4⁺ T cellhelp to OVA-specific CD8⁺ CTL by activating shared professional antigenpresenting cells (APCs) as suggested by many, and demonstrated recently(Bennett, S. R. M., et al., Nature, 393:478-480 (1998); Ridge, J. P., etal., Nature, 393:474-478 (1998) and Schoenberger, S. P., et al., Nature,393:480-483 (1998)).

As described herein, this cognate help hypothesis was tested using CD4deficient (knockout) mice (CD4−/−). Wild-type C57BL/6, CD4−/−, andβ2m−/− mice were each immunized with OVA or OVA.TBhsp70 fusion protein.As expected, immunization of wild-type mice with OVA.TBhsp70, but notOVA, generated CTL specific for the immunodominant epitope of OVA(SIINFEKL) (FIG. 1A). The same results were obtained when the CD4−/−mice were immunized with OVA.TBhsp70 (FIG. 1B). β2m−/− mice, which havevery few CD8⁺ T cells, did not develop OVA-specific CTL afterimmunization with OVA.TBhsp70 or with OVA alone (FIG. 1C).

Previous efforts to determine whether CD4⁺ T cell help is necessary forgeneration of CD8⁺ CTL have drawn differing conclusions. CD4 knockoutmice exhibit a range of CD8⁺ CTL responses: CD4-dependent, weaklydependent, or independent. CTL responses to minor histocompatibilityantigens (Ridge, J. P., et al., Nature, 393:474-478 (1998), Guerder, S.and Matzinger, P., J. Exp. Med., 176:553-564 (1992)) or to ovalbuminloaded into spleen cells (Bennett, S. R. M., et al., J. Exp. Med.,186:65-70 (1997)) are CD4-dependent. Some potent CD8⁺ T cell immunogensincluding viruses (Bachmann, M. F., et al., J. Immunol., 161:5791-5794(1988)), such as lymphocytic choriomeningitis virus (Leist, T. P., etal., J. Immunol., 138:2278-2281 (1987); Ahmed, R., et al., J. Virol.,62:2102-2106 (1988); Rahemtulla, A., et al., Nature, 353:180-184 (1991)and von Herrath, M., et al., J. Virol., 70:1072-1079 (1996)), ectromeliavirus (Buller, R. M., et al., Nature, 328:77-79 (1987)) and someinfluenza virus subtypes (Wu, Y. and Liu, Y., Curr. Biol., 4:499-505(1994)), as well as allogeneic cells (Krieger, N. R., et al., J. Exp.Med., 184:2013-2018 (1996)) elicit strong CD8⁺ T cell responses inwild-type and CD4−/− mice. The similarity of CD8 CTL responses toOVA.TBhsp70 in CD4−/− and wild-type mice suggests that hsp70 fusionproteins are relatively potent CD8⁺ CTL immunogens. A similar result,showing that CD4⁺ T cells are not required for the CD8⁺ CTL responseelicited by another mycobacterial heat shock fusion protein (hsp65 fusedto a polypeptide containing an epitope for 2C CD8⁺ T cells) using CD4−/−mice is described in Example 2. In addition, the ability of anon-homologous hsp, gp96, to elicit tumor rejection requires CD4⁺ Tcells at tumor challenge, but not during priming with tumor-derived gp96(Udono, H. and Srivastava, P. K., Proc. Natl. Acad. Sci. USA,91:3077-3081 (1994)).

It has been proposed that the immunostimulatory effects of certain hspfusion proteins may be due to the bacterial origin of the hsp moiety(Schild, H., et al., Curr. Opionon Imm., 11: 109-113 (1999)). Thispossibility was examined by making OVA.hsp70 fusion proteins with themurine homologue of TBhsp70 (Hunt, C. and Calderwood, S., Gene,87:199-204 (1990)), here referred to as mhsp70. Immunization ofwild-type C57BL/6 mice with OVA.mhsp70, but not OVA, elicited CTLresponses equivalent to those generated by the TBhsp70 fusion protein(FIG. 2A). The response to OVA.mhsp70 was also independent of CD4 (FIG.2B). Since a CD4⁺ T cell response to self (murine) hsp70 is unlikely,the effectiveness of the murine hsp70 fusion protein is in accord withthe more direct evidence for CD4-independence obtained using CD4−/− mice(see above).

The ability of hsp fusion proteins to elicit CTLs against the fusionpartner may be a consequence of the hsp moieties' chaperone activity,assuming that this activity is preserved in the fusion protein. Toinvestigate this issue, TBhsp70 was divided into four linear segmentsand OVA and a glycine/serine linker were fused to the amino-terminus ofeach segment, creating OVA.TBhsp70 s I-IV (FIG. 3). Each segmentcorresponds to a distinct structural domain of hsp70 as described byFlaherty, K. M., et al., Nature, 346:623-628 (1990) and Zhu, X., et al.,Science, 272:1606-1614 (1996). As shown in FIG. 3A, the amino-terminalATP-binding domain was divided into its two structural lobes: I (aa1-160) and II (aa 161-362). The carboxy-terminal peptide-binding domainwas divided into a β-sandwich domain, III (aa 364-512), and an α-helicaldomain, IV (aa 512-625).

Six groups of three C57BL/6 mice were immunized with 120 pmoles of OVA,OVA.TBhsp70, and OVA fused to segments I, II, III and IV. CTL assaysshowed that splenocytes from mice immunized with OVA.TBhsp70 and OVAfused to segment II lysed T2-K^(b) cells in the presence, but notabsence, of the OVA K^(b) epitope, SIINFEKL (FIG. 4). In contrast, cellsfrom mice immunized with OVA and OVA fused to segments I, III and IVwere ineffective, even at an E:T ratio of 80:1. Levels of cytolysisobtained with splenocytes from mice immunized with OVA.TBhsp70 and OVAfused to segment II were indistinguishable (FIG. 4). These results showthat half of the ATP-binding domain of TBhsp70 (aa 161-362) issufficient to stimulate substantial production of anti-OVA CTL responsein the absence of adjuvant.

Since it is highly unlikely that segment II preserves chaperone activitywe conclude that the ability of the fusion proteins to elicit CD8⁺ Tcell does not depend on the hsp moieties' chaperone properties. The datadescribed herein support a model in which hsp70 bypasses the need forCD4⁺ help by directly or indirectly activating or affecting thematuration state of APCs such as dendritic cells in a manner similar tosome viruses (Ruedl, C., et al., J. Exp. Med., 189:1875-1883 (1999)).According to this model, hsp70 fusion proteins likely activate few CD8⁺T cells to release immunostimulatory cytokines in draining lymph nodes.These cytokines, in turn, provide the help required to upregulateexpression of costimulatory molecules on APCs in the lymph node, leadingto further CD8⁺ T cell activation (Ruedl, C., et al., J. Exp. Med.,189:1875-1883 (1999)). Recent studies demonstrate that exposure ofmacrophages to bacterial and human hsp60 (Chen, W., et al., J. Immunol.,162:3212-3210 (1999); Kol, A., et al., J. Clin. Invest. 103:571-577(1990)), murine hsp70 and gp96 (Suto, R. and Srivastava, P. K., Science,269:1585-1588 (1995); Breloer, M., et al., J. Immunol., 162:3141-3147(1999)) increases expression of adhesion molecules and cytokines.

The ability to hsp70 fusion proteins to elicit CTL responses in theabsence of CD4⁺ cells indicates that hsp70 can be used as a vehicle forthe development of prophylaxis and therapy of diseases or conditionscharacterized by a lack or deficiency of CD4⁺ cells, such as HIV-1 andits opportunistic infections. Infections by HIV and its simian cousinSIV can lead to a substantial reduction of CD4⁺ T cells, therebycrippling the host's immune response to HIV and other pathogens. Thisloss of CD4⁺ cells is thought to impair the development and maintenanceof CD8⁺ CTL responses (Kalams, S. A., et al., J. Virol., 73:6715-6720(1999)). Recent studies conclude that strong HIV-specific CTL responsesare required to keep HIV-1 infection in check and to destroyHIV-infected cells (Harrer, T., et al., AIDS Res. Hum. Retro.,12:585-592 (1996); Harrer, T., et al., J. Immunol., 156:2616-2623(1996); Yang, O. O., et al., J. Virol., 70:5799-5806 (1996); Yang, O.O., et al., J. Virol., 71:3120-3128 (1997); Matano, T., et al., J.Virol., 72:164-169 (1998) and Wagner, L., et al., Nature, 391:908-911(1998)).

Example 2 Heat Shock Fusion Proteins Stimulate Dendritic Cells andElicit Production of Cytolytic T Lymphocytes Without RequiringParticipation of CD4 T Cells

Methods and Materials

Mice, CTL Clones and Cell Lines

C57BL/6 (H-2^(b)), Cd4-deficient (CD4 tm1Mak, H-2^(b)), and C3H/HeJ mice(H-2^(k)) were obtained from The Jackson Laboratories (Bar Harbor, Me.),maintained in barrier cages under specific pathogen free conditions, andimmunized between 4- and 10-weeks of age. 2C TCR transgenic mice(H-2^(b)) contain the rearranged transgenes encoding the αβ TCR from a2C CTL clone (Sha, W., et al., Nature, 335:271-274 (1988)). 2C TCRtransgenic mice deficient for the recombination activating gene-1(termed 2C/RAG) (Manning, T., et al., J. Immunol., 159:4665-4675 (1997))were used as a source of naive T cells for in vitro assays (Cho, B., etal., Proc. Natl. Acad. Sci. USA, 96:2976-2981 (1999)). 2C CTL clone.L3.100, has been previously described (Sykulev, Y., et al., Immunity,9:475-483 (1998)). EL4 cells were obtained from the ATCC (Rockville,Md.) and T2-K^(b) cells were a generous gift from Peter Creswell, YaleUniversity.

Plasmids, Peptides, and Proteins

In the P1 polypeptide the sequences flanking the—and C-termini of theSYRGL octapeptide (FIG. 5A), from ovalbumin (ova251-257) andα-ketoglutaraldehyde dehydrogenase, respectively, were modified byaddition of a lysine residue penultimate to the N-terminus (out ofubiquitination consideration) (Eisenlohr, L., et al., J. Exp. Med.,175:481-487 (1992); York, I. A. and Rock, K. L., Annu. Rev. Immunol.,14:369-396 (1996)), and an isoleucine and a tyrosine residue were addedat the—and C-termini for cloning purposes. Complementaryoligonucleotides encoding P1 were synthesized and cloned into amammalian expression vector VR1055 (Vical, San Diego, Calif.), andsubsequently subcloned as an in-frame fusion at the 3′ end of M. bovisBCG hsp65 gene (hsp65-P1) into the bacterial expression vector pET28A+(Novagen, Madison, Wis.). The P1 sequence was also subcloned into the 3′end of the gene encoding E. coli maltose binding protein in pMAL-p2,using the pMAL protein fusion system (New England Biolabs, Beverly,Mass.), as well as into the mammalian expression vector pClneo (Promega,Madison, Wis.). All hsp65 fusion proteins used in this example, as wellas the unmodified hsp65, were produced as recombinant proteins in Ecoli. They were purified under denaturing conditions from the solublefraction of bacterial lysates and fractionated successively onbutyl-Sepharose, Q-Sepharose (and Ni-Sepharose when applicable), andfinally by dialysis against PBS. Ma1-P1 was purified by amylose affinitychromatography (New England Biolabs, Beverly, Mass.).

SDS-PAGE analysis of purified hsp65-P1 revealed a major species at 67.5kDa, which was shown to be hsp65-P1 by Western analysis, usinganti-mycobacterial hsp65 specific antibody (StressGen, Victoria,Canada), and by electrospray mass spectrometry (M.I.T. BiopolymerLaboratory). Ma1-P1 was also subjected to amino acid analysis andSDS-PAGE to confirm molecular weight (48.1 kDa) and purity. P1 and SYRGLpeptides were synthesized by the MIT Biopolymers Laboratory. Proteinconcentrations were estimated by bicinchoninic acid or amino acidanalyses and were expressed in molar terms to facilitate comparisonsbetween proteins and polypeptides of differing molecular masses.Endotoxin concentrations of recombinant protein preparations weredetermined by the Limulus assay, using reagents and conditions accordingto Associates of Cape Cod (Falmouth, Mass.). Peptide concentrations wereestimated by W/V or based on amino acid analyses.

Antibodies and Flow Cytometry

Flow Cytometry was carried out on a FACSCaliber, using CellQuestsoftware (Becton Dickinson, Franklin Lakes, N.J.). Unlabeled or FITC-,PE-, allophycocyanin- or biotin-labeled antibodies against CD69, CD4,CD8, CD11c, CD11b, GR.1, B7.2, B220 or MCH class I (H-2^(b)), as well assecondary antibodies and streptavidin labeled with allophycocyanin, orPE, were obtained from Pharmingen (San Diego, Calif.). 1B2, a clonotypicantibody that recognized 2C TCR (Kranz, D., et al., Proc. Natl. Acad.Sci. USA, 81:573-577 (1984)), was purified from the 1B2 hybridoma andbiotinylated using biotinamidocaproate N-hydroxysuccinimide ester(Sigma, St. Louis, Mo.). The antibody, Y3, is cross-reactive with MCHclass I from H-2^(b) (K^(b)) and H-2^(k) haplotypes. It is affinitypurified from culture supernatants from the Y3 hybridoma (obtained fromATCC, Rockville, Md.) and labeled with fluorescein using fluoresceinisothiocyanate.

Generation of Bone-marrow Derived Dendritic Cells and Isolation ofAntigen Presenting Cells and Naive 2C T Cells

To generate bone-marrow derived dendritic cells from C57BL/6 (or C3H)mice, bone marrow was flushed from the femur and tibia, red blood cellswere lysed, and the remaining cells were cultured at 10⁶ cells/ml inRPMI 1640 medium (supplemented with 10% heat-inactivated fetal calfserum, 2 mM L-glutamine, 10 mM HEPES, 50 μm β-mercaptoethanol, 100 U/mlpenicillin and 100 μg/ml streptomycin) containing 20 ng/ml murine GM-CSF(R&D Systems, Minneapolis, Minn.). The medium was replaced on days 2 and4, and on day 6 the cells (immature dendritic cells) were harvested foruse.

In vitro assays were performed with purified cell populations unlessotherwise noted. Magnetic cell sorting (MACS) was carried out accordingto the manufacturer's instructions (Miltenyi Biotec. Auburn, Calif.).Dendritic cells (splenic or bone marrow-derived) were isolated bypositive sorting using anti-CD11c antibody (purity ranged from 70-97%).Peritoneal lavage macrophages were purified by treating them withbiotinylated antibodies specific for CD11c, GR.1, and B220, followed bywashing and incubating them with magnetic microbeads coated withanti-CD4 or anti-CD8 antibodies or with streptavidin and then passingthem over a negative sorting column. Macrophage purity wastypically >90%. For purification of 2C T cells from 2C/RAG transgenicmice, lymph node and spleen cells were coated with anti-CD8 magneticbeads (an average of 2 beads per cell) and positively sorted as above(purity >93%). The purification procedures did not activate APC or Tcells as shown by flow cytometry: APC showed no increase in B7.2, MHCclass I, or cell diameter, and T cells showed no CD69 upregulation after24 hr in culture. There was also no significant ³H-thymidineincorporation by T cells after 48 hr incubation.

Cytolytic T Cell Assays

Unless otherwise noted, ⁵¹Cr-labeled T2-K^(b) cells were used as targetcells. They were incubated with effector cells derived from eitherfusion protein-injected mice or from cultured 2C T cell clones for 4 hrin the presence or absence of SYRGL (1 μM). Specific lysis wascalculated as follows: [(experimental counts−spontaneous counts)/(totalcounts−spontaneous counts)]×100.

To assess the ability of various APC to process hsp65-P1, dendriticcells and macrophages were used as target cells. Each of these cellpopulations was purified by MACS and then ⁵¹Cr-labeled for 1 hr at 37°C. The labeled cells were then incubated with hsp65-P1 together with the2C CTL clone (L3.100) at a CTL: target cell (E:T) ration of 5:1. Assayswere performed in triplicate using 96-well round bottom plates and cellsupernatants were counted in a γ spectrometer after 4 hr. Specific lysiswas calculated as above.

Transient Transfection and Antigen Processing Assays

EL4 cells (5×10⁶) were electroporated with 15 μg of the parent plasmidsor plasmids containing the genes for P1 (in VR1055) or hsp65-P1 (inpClneo). 48 hr after transfection, the cells were subjected tocentrifugation in Ficoll-Paque (Pharmacia Biotech., Piscataway, N.J.)(2200rpm, 20 min) and 10⁶ live cells were incubated with an equal numberof splenocytes from naive 2C/RAG mice. After 18 hr the cells werestained with 1B2, anti-CD69, and anti-CD8 antibodies (labeled with FITC,PE, and allophycocyanin, respectively) and 2C T cells were evaluated forupregulation of CD69 by flow cytometry, gating on propidiumiodide-negative, 1B2⁺, CD8⁺ cells.

Naive 2C T Cell Responses to Dendritic Cells and Macrophages andDendritic Cell Activation Assays

Purified dendritic cells and macrophages were incubated with variousconcentrations of proteins or peptides in 96-well (5×10⁴ cells/well)flat bottom plates for 24 hr at 37° C. The following day an equal numberof purified naive 2C T cells were added to each well (final volume: 200μl for the 96-well plates, 600 μl for the 48-well plates). After 18 hr.,the 48-well plates were separated into i) cell pellets to analyze 2C Tcells for expression of the acute activation marker CD69 by flowcytometry, gating on propidium iodide-negative. 1B2⁺ CD8⁺ cells, and ii)cell supernatants to measure IL-2 secretion (in triplicate, using HT2cells in a standard bioassay) (Watson, J., J. Exp. Med., 150:1510-1519(1979)). After 48 hr, the 96-well plates were assayed for IFN-γsecretion (using 50 μl of cell supernatants and a capture ELISA assay(R&D Systems, Minneapolis, Minn.), and for T cell proliferation (1 mCi³H-thymidine (NEN, Boston, Mass.) was added per well and 16 hr later thecells were harvested to measure ³H-thymidine-incorporation). Whereindicated, 1B2 Fab fragments were added to naive 2C T cells at a finalconcentration of 25 μg/ml.

Immature bone marrow-derived dendritic cells (day 6 of culture) werepurified by magnetic sorting (>95% CD11b⁺CD11c⁺) and incubated (2.5×10⁵cells/well in 96-well round bottomed plates) with various fusionproteins or control proteins. The following day, cells were analyzed byflow cytometry for expression of B7.2 and MHC class I and class IImolecules, gating on propidium iodide-negative, CD11c⁺ cells.

RESULTS

Design and Characterization of Heat Shock Fusion Protein hsp65-P1

As shown in FIGS. 5A and 5B, the principal fusion protein used hereincontains the polypeptide P1 fused to the C-terminus of hsp65. P1includes the octapeptide, SYRGL, that behaves, in association withK^(b), as a strong agonist for the TCR on 2C T cells (Sykulev, Y., etal., Immunity, 4:565-571 (1996)). The sequences that flank theoctapeptide in P1 were chosen because they correspond to those known tobe effectively cleaved intracellularly in two unrelated proteins:ovalbumin (Falk, K., et al., Eur. J. Immunol., 22:1323-1326 (1992)) andα-ketoglutaraldehyde dehydrogenase (Udaka, K., et al., Cell, 69:989-998(1992), see arrows, FIG. 5A). To determine if the P1 polypeptide, aloneor linked as a fusion partner to hsp65, could be cleaved intracellularlyto liberate the SYRGL octapeptide, we transfected plasmids containingsequences for P1 or hsp65-P1 were transfected into EL4 cells (H-2^(b)).Because relatively few of the transiently transfected cells wereexpected to express P1 or hsp65-P1, the transected cell population wasnot used in cytolytic assays as targets for 2C CTL. Instead, theirability to stimulate naive 2C T cells were examined. As shown in FIG.5C, 80-90% of these naive T cells were stimulated to express the acuteactivation marker CD69 in response to EL4 cells transfected with eitherthe P1 or hsp65-P1 plasmids, while virtually none of the naive T cellswere activated by cells transfected with the empty plasmids (vector,shaded histograms, FIG. 5C). These results indicate that in thesetransfected cells P1 and hsp65-P1 can be cleaved to release theoctapeptide, which is then presented by K^(b). C57BL/6 mice produceSYRGL-specific CD8⁺ cytolytic T cells in response to hsp65-P1

Before immunizing mice with hsp65-P1, it was first ensured that CD8 Tcells that can recognize the SYRGL octapeptide (SEQ ID NO: 4) arepresent in normal C57BL/6 (H-2^(b)) mice. The mice were thereforeinjected with SYRGL peptide (SEQ ID NO: 4) in adjuvant (TiterMaxGold),their spleen cells were maintained in culture for several weeks (seeMethods) and subsequently tested in a standard cytotoxicity assay. Asshown in FIG. 5D, the cell line's lysis of K^(b+) target cells(T2-K^(b)) was SYRGL (SEQ ID NO: 4)-dependent, indicating the presencein these mice of T cells that can respond to SYRGL-K^(b) complexes.

To determine if the hsp65-P1 fusion protein could stimulate (“prime”)anti-SYGRL CTL in vivo, normal C57BL/6 mice were injected subcutaneously(s.c.) with the fusion protein in saline without added adjuvants. Eachmouse received two injections, one wk apart. 7 days after the 2^(nd)injection cells from regional lymph nodes and spleen were restimulatedin culture with SYRGL (1 μM) in the absence of exogenous cytokines, andtested after 6 days for CTL activity in a 4 hr cytolytic assay, using⁵¹-Cr labeled K^(b+) target cells (T2-K^(b); see Methods). Of 40injected mice, 35 produced CTL whose lysis of the K^(b+) target cellswas SYRGL-dependent (see FIG. 6A for a representative response). C57BL/6mice treated in exactly the same way with equimolar amounts of variouscontrols (hsp65, P1, or a mixture of hsp65 and P1, or SYRGL alone), allfailed to yield SYRGL-specific CTL (FIG. 6A). As little as 1 μg (0.015nmoles) of hsp65-P1 could elicit an anti-SYRGL CTL response. A controlfusion protein, made by fusing the P1 sequence to the C-terminus ofanother bacterial protein chosen simply for ease of purification (the E.coli maltose binding protein), here called Ma1-P1, was around 10-100times less effective in these assays (FIG. 6B) and without anydetectable effect in others (FIGS. 7, 8A-8C, 9A-9C, 10A-10C). Removal ofCD8 T cells by magnetic sorting showed that the cytolytic response tohsp65-P1 was due to CD8 T cells (FIG. 6C). These results demonstratethat hsp65-P1, without added adjuvants, can elicit a CD8 T cell responseto the fusion partner.

Dendritic Cells and Macrophages Differ in Ability to Serve asAntigen-presenting Cells for hsp65-P1

To identify antigen presenting cells (APC) that mediate in vivo CD8 Tcell responses, purified preparations of APC from C57BL/6 mice(dendritic cells from spleen or bone marrow, and macrophages fromperitoneal lavage) were tested for ability to present processed hsp65-P1and serve as target cells in cytolytic assays, using a well-establishedSYRGL-K^(b) specific CTL clone (L3.100) as effectors. When dendriticcells and macrophages were ⁵¹Cr-labelled and incubated with hsp65-P1 for4 hr, they were lysed effectively and to about the same extent (FIG. 7).No significant lysis was observed, however, when the control fusionprotein Ma1-P1 was used in place of hsp65-P1, suggesting that processingof hsp65-P1 by these APC was not due to indiscriminate extracellularproteolysis.

Cytolytic reactions with potent CTL clones, such as L3.100, can beexquisitely sensitive, detecting very few and probably as little as onecognate peptide-MHC complex per target cell, (Sykulev, Y., et al.,Immunity, 4:565-571 (1996)). Therefore, a more discriminating assay inwhich dendritic cells and macrophages that had been incubated withhsp65-P1 were compared as APC for their ability to stimulate naive 2C Tcells was used. As shown in FIG. 8A-8C, when the dendritic cells wereincubated with hsp65-P1 overnight and then with naive 2C T cells, thenaive T cells were stimulated to: i) express CD69, ii) proliferate, andiii) secrete IL-2 and IFN-γ. In contrast, the macrophage preparationsstimulated none of these responses. (It may be that activatedmacrophages would have behaved differently, but we deliberately focusedon non-activated macrophages and dendritic cells to stimulate conditionsin the immunized animal were deliberately focused upon. The responseelicited by dendritic cells could be inhibited by the clonotypic,anti-2C TCR, antibody (1B2; FIG. 8C), indicating that they were mediatedby ligation of the 2C TCR. The requirement for the hsp65 moiety in thehsp65-P1 fusion protein is emphasized by the result that naive 2C Tcells were stimulated to express CD69 by dendritic cells that had beenincubated with hsp65-P1 but not by those that had been incubated withthe control fusion protein Ma1-P1 (FIG. 8A).

Incubation of dendritic cells with various controls (P1 alone, hsp65alone, or a mixture of hsp65 ⁺P1) in place of hsp65-P1 did not stimulate2C T cells to secrete IFN-γ. However, of all the controls the P1 peptidewas exceptional in that it exhibited some activity; with both dendriticcells and macrophages it stimulated CD69 expression and with dendriticcells, but not with macrophages, it induced proliferation and IL-2secretion by the naive 2C T cells. It is likely that the P1 peptideitself is subject to proteolysis by these APC, particularly by dendriticcells, but whether extracellularly or in some intracellular compartmentis not clear. Whatever the explanation, it should be noted that the P1polypeptide did not stimulate CD8 CTL production in vivo underconditions where the hsp65-P1 fusion protein was consistently effective(FIG. 6A). In addition, as is shown later, P1 also failed to activatedendritic cells (see FIGS. 10A-10C).

To examine the difference between dendritic cells and macrophages moreclosely, these cells were incubated with various concentrations of thefusion proteins and then evaluated their ability to stimulate naive 2C Tcells. As shown in FIG. 9A, the naive cells proliferated and producedsubstantial amounts of IL-2 in response to dendritic cells that had beenincubated with concentrations of hsp65-P1 in the 0.1-0.01 μM range. Incontrast, the responses by naive cells were negligible when macrophageswere used in place of dendritic cells or when the Ma1-P1 control fusionprotein was used at concentrations up to 1 μM (FIG. 9A). Togther, thesedata suggest that dendritic cells are more effective than macrophages inprocessing and presenting the octapeptide from hsp65-P1.

Heat Shock Fusion Proteins Stimulate Dendritic Cells Directly

The distinctive ability of hsp65-P1 to stimulate naive 2C (anti-SYRGL) Tcells in vitro only in the presence of dendritic cells led to theexamination of the effect of hsp65-P1 on dendritic cells directly. Asshown in FIG. 10A, when immature bone-marrow derived dendritic cells(day 6 in culture) were incubated overnight with various concentrationsof hsp65-P1 the dendritic cell surface level of an MHC class I molecule(K^(b)) was increased. The extent of the increase depended on thehsp65-P1 concentration, and no increase was seen when hsp65-P1 wasreplaced by a series of control proteins and peptides (hsp65 alone, P1alone, SYRGL, Ma1-P1, or a monoclonal IgG antibody [anti-2,4,6,trinitrophenyl]).

Other hsp65 fusion proteins, having various fusion partners (influenzavirus nucleoprotein or the E7 subunit of human papilloma virus) alsoelicited increased expression of K^(b) on the dendritic cells (FIG.10A). It is important to note, however, that unmodified hsp65 (“hsp65only” in FIG. 10A, 10B) consistently failed to stimulate dendritic cellupregulation of K^(b).

All of the fusion proteins as well as unmodified hsp65 were produced asrecombinant proteins in E. coli and contained trace levels of endotoxin(lipopolysaccharide, LPS). An endotoxin standard by itself evoked a weakresponse at the highest concentration tested (5 EU/ml, FIG. 10B).Because of mol. wt. heterogeneity of LPS, conversion of endotoxin unitsinto LPS weight and mole units is highly approximate. But, if one EUcorresponds to about 5 ng LPS, and the “average” mol. wt. of LPS istaken to be approximately 10,000, LPS would appear to be somewhat moreeffective than hsp65 fusion proteins in activating dendritic cells.Nevertheless, the effects of the fusion proteins seemed clearly not tobe due to endotoxin contaminants, because when hsp65-P1, hsp65, orMa1-P1 were each added in amounts that resulted in addition ofequivalent EU units to the dendritic cells, increased expression ofK^(b) was elicited only by hsp65-P1. Moreover, when the data from FIG.10A were plotted against the EU concentrations attributable to thecontrols and fusion proteins, it was evident that each of the four hsp65fusion protein preparations, but none of the controls, stimulatedincreased expression of MHC class I protein. Finally, all the hsp65fusion proteins elicited increases in MHC class I expression ondendritic cells from C3H/HeJ mice, a strain known to be unresponsive toLPS (due to a mutation in the Toll4 receptor) (Poltorak, A., et al.,Science, 282:2085-2088 (1998)). Taken together, the findings demonstratethat activation of the dendritic cells was due to the hsp65 fusionproteins, not to endotoxin contaminants. Besides stimulating thedendritic cells (bone marrow derived and maintained in culture withGM-CSF for 6 days) to express increased levels of MHC class I, the hspfusion proteins stimulated increased expression of MHC class II and B7.2(CD86) (the Table); the level of CD40 was, however, only marginallyaffected. Native hsp65 did not affect expression of MHC class II orB7.2, just as it failed to affect levels of MHC class I. TABLE Heatshock fusion proteins stimulate increased expression of MHC andcostimulatory (B7.2) molecules on dendritic cells unmodi- fied Nothinghsp65 P1 peptide hsp65-NP hsp65-P1 MHC Class I 100 105 96 257 151 MHCClass II 63 67 55 279 90 B7.2 60 54 47 129 81 CD40 45 45 47 76 51Dendritic cells from bone marrow of C3H/HeJ mice were incubated for 18hrs with 1.5×10⁻⁶M of various heat shock proteins or the control P1peptide prior to cell surface staining.

Activated Dendritic Cells in Vivo

That the dendritic cell changes could also be elicited in vivo wasindicated by the finding that 24 hrs after injecting hsp65-P1 (insaline) subcutaneously into mice, myeloid dendritic cells (but notlymphoid dendritic cells) from lymph nodes draining the site ofinjection showed increased expression of K^(b) (FIG. 9B).

To determine if activated DC were especially effective in vivo, normalB6 mice were adoptively transferred with 2×10⁶ naive 2C cells (from 2CTCR transgenic mice, see Cho, B., et al., Proc. Natl. Acad. Sci. USA,96:2976-2981 (1999)) and the next day the recipients were injected in ahind footpad with 8×10⁵ dendritic cells. The dendritic cells had beenincubated overnight with or without hsp65-NP (to generate activated ornonactivated dendritic cells, respectively), and then incubated for 2hrs with SYRGL peptide at various concentrations (0, 10⁻⁶, 10³¹ ⁷, 10⁻⁸,10⁻⁹M) and washed just before the cells were injected. 24 hrs later 2CCD8⁺ T cells from the draining popliteal lymph nodes were examined forCD69 expression as evidence of having been antigenically stimulated. Asshown in FIG. 9C, when the peptide concentration was 10⁻⁹M, theactivated dendritic cells were considerably more effective than thenonactivated dendritic cells in stimulating the naive 2C T cells toexpress CD69. When pulsed with the peptide at 10-1000-times higherconcentrations activated and nonactivated dendritic cells were aboutequally effective, stimulating CD69 responses of the 2C CD8⁺ cells atabout the level seen in FIG. 9C, bottom panel.

Stimulation of CD8 CTL Production in vivo by the hsp65-P1 Fusion ProteinDoes Not Require the Participation of CD4 T Cells

The ability of the hsp fusion proteins to directly stimulate dendriticcells suggested that CD4 T cells might not be necessary for the CD8 Tcell response elicited in vivo by the fusion proteins. To test thispossibility, CD4 knockout mice (CD4^(−/−)) were immunized using the sameregimen as before (FIG. 6A-6C) and their ability to produceSYRGL-specific CTL was assessed. As seen in a representative response inFIG. 10C, the CD4^(−/−) mice produced CTL in response to hsp65-P1 butnot in response to the control Ma1-P1. While the cytolytic activityelicited in the CD4^(−/−) mice (n=6) was unambiguous, it appeared to besomewhat less than was generally elicited in normal C57BL/6 mice. Inother experiments C57/BL6 mice that had been extensively depleted of CD4cells by repeated injections or an anti-CD4 mAb (GK1.5) also respondedto the standard immunization protocol with hsp65-P1 about as well asuntreated normal mice (data not shown). All of these results show thatin stimulating CD8 CTL production in mice hsp65-P1 does not require theparticipation of CD4 T cells.

Discussion

As shown herein, mycobacterial hsp65 fused to the P1 polypeptideactivates dendritic cells and stimulates, in the absence of CD4⁺ Tcells, the production of CD8⁺ CTL that recognize a short peptide derivedfrom P1. The findings extend the number and diversity of hsp fusionproteins that can elicit CD8 T cell responses and suggest a potentialmechanism by which the fusion proteins exert their effects in theabsence of added adjuvants, a prominent feature of the in vivo responsesto all hsp fusion proteins. Generally, where CD4 T cell help andadjuvants are required for CD8 T cell responses, it is likely that theyfunction by activating dendritic cells (Bennett, S., et al., Eur. J.Immunol., Nature, 393:478-480 (1998); Ridge, J., et al., Nature,393:474-478 (1998) and Schoenberger, S., et al., Nature, 393:480-483(1998)). It is reasonable to expect that the capacity of heat shockfusion proteins to directly activate dendritic cells accounts for theirability to bypass the requirements for CD4 T cells and added adjuvants.

HSP Fusion Proteins Activate Dendritic Cells Directly

Using CTL to detect polypeptide processing by APC, previous studiespointed to macrophages, or equally to macrophage and dendritic cells, asbeing responsible for processing protein immunogens that elicit CD8 Tcell responses (Kovacsovics-Bankowski, M., et al., Science, 267:243-245(1995); Rock, K., Today, 17:131-137 (1996); Suto, R. and Srivastava, P.K., Science, 269:1585-1588 (1995)). As shown herein, when macrophagesand dendritic cells were incubated with hsp65-P1 they become equallysusceptible to lysis by peptide-specific CTL in a standard 4 hrcytolytic assays, indicating that both types of APC could generate smallpeptides from the hsp fusion protein and load them on the MHC class Imolecules. However, when these cells were evaluated for their ability tostimulate naive CD8 T cells to proliferate and produce IL-2 and IFN-γ,the dendritic cells, but not the macrophages, proved to be effective. Astep towards understanding this difference comes form the presentfinding that hsp65-P1, as well as each of the other hsp65 fusionproteins tested, is capable of directly stimulating dendritic cells toincrease their surface expression of MHC class I and II andcostimulatory (B7.2) molecules.

Dendritic cells infected with mycobacteria, including BCG, orstreptococci, or Leishmania have been shown to upregulate MHC andcostimulatory molecules B7.1 and B7.2 and, in addition, to secrete IL-12(Demangel, C., et al., Eur. J. Immunol., 29:1972-1979 (1999); Henderson,R., et al., J. Immunol., 159:635-643 (1997); Konecny, P., et al., Eur.J. Immunol., 29:1803-1811 (1999); Rescigno, M., et al., Proc. Natl.Acad. Sci. USA, 95:5229-5234 (1998)). It may be that microbial cell hspmolecules are responsible for these effects. If so, the findingsdescribed herein (of a difference between hsp65 fusion proteins andunmodified hsp65) indicate that upregulation of these activationmolecules are due to the hsp in a modified form, resembling perhaps thehsp65 fusion proteins studied here, rather than naive hsp molecules.

Dendritic cells infected with certain viruses, e.g., influenza virus(Ridge, J., et al., Nature, 393:474-478 (1998)), likewise becomeactivated. However, the hsp fusion proteins appear, so far, to be theonly soluble immunogenic proteins that directly activate dendriticcells, in vitro and in vivo, to upregulate expression of MHC andcostimulatory molecules. The experimental system described herein isuseful for investigating the pathways by which hsp fusion proteins areprocessed and presented by dendritic cells and the mechanisms by whichMHC and costimulatory molecules are up-regulated.

Hsp Fusion Protein Stimulation of CD8 T Cell Production Does Not DependUpon CD4 T Cells

Prior to the present study one way to account for the ability of hspfusion proteins to stimulate CD8 T cell production was to invoke a keyrole for CD4 T cells. Thus, a vigorous CD4 T cell response to peptidesfrom the hsp moiety could activate dendritic cells and amplify anotherwise marginal CD8 T cell response to peptides from the fusionpartner (Bennett, S., et al. Nature, 393:478-480 (1998); Ridge, J., etal., Nature, 393:474-478 (1998); Schoenberger, S., et al., Nature,393:480-483 (1998)). This possibility is supported by older evidencethat hsp65 can serve as an effective carrier molecule in the classicsense: i.e., when chemically coupled to nonimmunogenic hapten-likemolecules (polysaccharides, a malarial peptide) the conjugates elicitedIgG antibodies to the adducts in responses that were presumably T-celldependent (Barrios, C., et al., Eur. J. Immunol., 22:1365-1372 (1992)).This mechanism is clearly not essential, because CD4^(−/−) mice injectedwith hsp65-P1 produced cytolytic CD8 T cells to the fusion partner'speptide. Nevertheless, it is entirely possible that in normal animalsthe response may be enhanced by CD4 T cells specific for peptidesderived from the hsp moiety.

Previous efforts to determine whether CD4 T cells are essential for CD8T cell responses to various immunogens and immunization strategies haveyielded diverse results. With some epitopes, e.g., minorhistocompatibility antigens, CD8 T cell responses could not be elicitedin CD4^(−/−) mice (DiRosa, F. and Matzinger, P., J. Exp. Med.,183:2153-2163 (1996)), but with more potent immunogens, (e.g.,lymphocytic choriomeningitis virus or a murine herpes virus), or highdoses of particulate antigens, CD8 CTL responses in CD4^(−/−) mice werevirtually the same as in normal mice (Rahemtulla, A., et al., Nature,353:180-184 (1991); Rock, K. and Clark, K., J. Immunol., 156:3721-3726(1996); Stevenson, P., et al., Proc. Natl. Acad. Sci. USA,95:15565-15570 (1998)). That CD4 T cells are not required for the CD8 Tcell response to hsp65-P1 indicates that hsp fusion proteins arerelatively potent immunogens for CD8 T cells.

The hsp Moiety in hsp Fusion Proteins

In the several hsp fusion proteins examined here the only common elementis hsp65. The question arises as to how the hsp moiety can directlyactivate dendritic cells (and thereby elicit CD8 CTL production),regardless of wide variations in length and sequence of the fusionpartners. It is particularly notable, in contrast, that unmodified(“native”) hsp65 lacks this critical activity. It may be that in thefusion proteins the hsp moiety adopts a particular conformation ordisplays a linear sequence or peptide motif or pattern that is i)necessary for eliciting the dendritic cell response, ii) retaineddespite wide variations in the fusion partner sequences, and iii) absentor masked in unmodified (“native”) hsp65.

The intensity of current interest in CD8 vaccines for HIV-1 and otherpersistent intracellular pathogens, as well as for cancer cells, isreflected in recent studies of diverse genetic vaccines and of severalbacterial toxins fused to antigenic peptides or polypeptides asstimulators of CD8 CTL production. For example, nonapeptide sequencesinserted into a truncated subunit of anthrax toxin or pertussis toxin,could elicit CD8 CTL in vivo (Ballard, J., et al., Proc. Natl. Acad.Sci. USA, 93:12531-12534 (1996) and render target cells susceptible tolysis by cognate CD8 CTL in vitro (Goletz, T., et al., Proc. Natl. Acad.Sci. USA, 94:12059-12064 (1997); Carbonetti, N., et al., Infect. Immun.,67:602-607 (1999)). These and other bacterial toxins have evidentlyacquired through evolution the capacity to cross mammalian cellmembranes and gain access to the cell cytosol, where they exert theirlethal effects. While the judicious linkage of small peptides allowsthese toxin subunits to retain their ability to traverse membranes, theneed to preserve this special property may limit the size and sequencediversity of the fusion elements that can be accommodated. For the hspfusion proteins, in contrast, there appear so far to be no constraintsto their effectiveness as CD8 immunogens by the length or sequence ofthe fusion partners. Because large fusion partners, e.g., the equivalentof a typical protein domain, are likely to encompass many potentialepitopes for diverse MHC class I molecules, the hsp fusion proteins as aclass are candidate vaccines for use with populations of MHC-disparateindividuals.

While this invention has been particularly shown and described withreferences to preferred embodiments thereof, it will be understood bythose skilled in the art that various changes in form and details may bemade therein without departing from the scope of the inventionencompassed by the appended claims.

1. A method of inducing an immune response that includes a CD8⁺cytotoxic T lymphocyte (CTL) response to a molecule in an individual,the method comprising administering to the individual the moleculejoined to a heat shock protein or the molecule joined to anadenosinetriphosphate (ATP) binding domain of a heat shock protein or aportion thereof.
 2. The method of claim 1, wherein the individual has adeficiency of CD4⁺ T cells.
 3. The method of claim 1, wherein the heatshock protein is fused to the molecule.
 4. The method of claim 1,wherein the ATP binding domain is fused to the molecule.
 5. The methodof claim 1, wherein the heat shock protein is covalently bonded orchemically conjugated to the molecule.
 6. The method of claim 1, whereinthe ATP binding domain, or the portion thereof, is covalently bonded orchemically conjugated to the molecule.
 7. The method of claim 1, whereinthe molecule is a protein or glycoprotein.
 8. The method of claim 1,wherein the molecule is a carbohydrate or lipid.
 9. The method of claim1, wherein the molecule is a bacterial or viral antigen.
 10. The methodof claim 1, wherein the viral antigen is an antigen of the humanimmunodeficiency virus.
 11. The method of claim 1, wherein the moleculeis a parasitic antigen.
 12. The method of claim 1, wherein the moleculeis a cancer cell-associated antigen.
 13. The method of claim 1, whereinthe heat shock protein, the ATP binding domain of the heat shockprotein, or the portion thereof, is a mycobacterial protein.
 14. Themethod of claim 13, wherein the mycobacterial protein is an M. leprae,M. bovis, or M. tuberculosis protein.
 15. The method of claim 1, whereinthe heat shock protein, the ATP binding domain of the heat shockprotein, or the portion thereof, is hsp65, hsp70, or hsp90.
 16. Themethod of claim 1, wherein the heat shock protein, the ATP bindingdomain of the heat shock protein, or the portion thereof is a mammalianprotein.
 17. The method of claim 16, wherein the mammalian protein is ahuman protein.
 18. The method of claim 1, wherein the portion of the ATPbinding domain consists of about half of the ATP binding domain.
 19. Themethod of claim 1, wherein the portion of the ATP binding domain is aportion of a naturally occurring ATP binding domain in which 1-50% ofthe amino acid residues have been substituted; 10-40% of the amino acidresidues have been substituted; or 10-20% of the amino acid residueshave been substituted.
 20. The method of claim 19, wherein at least halfof the substituted amino acid residues are conservative amino acidsubstitutions.
 21. The method of claim 1, wherein the portion of the ATPbinding domain comprises amino acid residues 161-370 of Mycobacteriumtuberculosis hsp70.
 22. The method of claim 2, wherein the individualhas an acquired immune deficiency syndrome.
 23. A method of inducing aCD4⁺-independent cytotoxic T lymphocyte response to a molecule in anindividual, the method comprising administering to the individual aportion of an ATP binding domain of a heat shock protein joined to themolecule.
 24. The method of claim 23, wherein the molecule is a protein,a peptide, a glycoprotein, a carbohydrate, a viral antigen, a fungalantigen, or a parasitic antigen.
 25. The method of claim 23, wherein theheat shock protein is an hsp65, hsp70, hsp90, bacterial, mycobacterial,fungal, parasitic, or mammalian heat shock protein.